Effects of nitrate and ammonium on assimilation of nitric oxide by Heterosigma akashiwo

The harmful alga Heterosigma akashiwo possesses a hybrid nitrate reductase (NR) enzyme, NR2-2/2HbN, which has the potential to convert NO to nitrate for assimilation into biomass. In previous research, NR transcription in H. akashiwo was induced by nitrate while NR activity was inhibited by ammonium. Here, the capacity of H. akashiwo to use NO in the presence of nitrate and/or ammonium was investigated to understand the regulation of NO assimilation. Continuous cultures of H. akashiwo were acclimated to growth on nitrate, ammonium, or a mixture of both. Aliquots from these cultures were spiked with 15N-labeled NO. The expression of genes involved in nitrogen assimilation was evaluated, as well as nitrate reductase activity and assimilation of 15N-labeled nitrogen into algal biomass. Results showed that NO induced expression and activity of NR, and upregulated expression of GOGAT regardless of the presence of other inorganic nitrogen sources, while GS expression decreased over time. Furthermore, 15NO uptake and assimilation was significantly higher in cultures acclimated for growth on ammonium compared to cultures acclimated for growth on nitrate alone. Assimilation of NO may provide H. akashiwo with a competitive advantage in N-poor environments or areas with elevated NO.

www.nature.com/scientificreports/ and activity is upregulated by nitrate and repressed by nitrite and ammonium as well as downstream products of the GS/GOGAT pathway 22 .
In contrast to prototypical NR, however, NR2 in H. akashiwo includes a truncated hemoglobin (HbN) domain 9 . Similarities to mycobacterial HbN 23 and truncated hemoglobins in other algal species 24 suggest that NR2 converts NO to nitrate via nitric oxide dioxygenase (NOD) activity of the HbN domain 9 , which may then be reduced to nitrite via NR activity. In support of this hypothesis, NR in Chlamydomonas has been shown to associate with truncated hemoglobin (THB1) to convert NO to nitrate 24 . However, NR also participates in generation of NO in Chlamydomonas through partnership with amidoxime-reducing component/nitric oxide-forming nitrite reductase (ARC/NOFNiR) 25 . The generation of NO through this pathway plays a role in repression of NR by ammonium, and has been shown to repress NR transcription and enzyme activity as well as nitrate transport when both ammonium and nitrate are present 26,27 . Assimilation of externally sourced NO through NOD activity of NR2 in H. akashiwo may be inhibited in a similar manner, effectively preventing H. akashiwo from accessing this nutrient when ammonium is present.
The object of this study was to investigate the regulation of NO assimilation into biomass by H. akashiwo, and the effects of other nitrogen sources on this process. Specifically, this study tested the hypothesis that the assimilation of NO is repressed in the presence of ammonium. This may affect the ability of H. akashiwo to access this nitrogen source in coastal environments impacted by ammonium pollution, and limit the role of NO in bloom initiation and maintenance.

Results
For this investigation, Heterosigma akashiwo was acclimated to growth on nitrate (Experiment 1), a 50/50 mix of nitrate and ammonium (Experiment 2) or ammonium alone (Experiment 3) (see Fig. 1). Subcultures from each experiment were spiked with 15 NO. Several parameters were measured to evaluate the response of H. akashiwo to addition of NO, including transcription of genes involved in nitrogen assimilation, NR enzyme activity, and assimilation of 15 N-labelled NO into particulate nitrogen. Paired replicates also received either nitrate or ammonium at 10% ambient concentrations instead of NO as a means to verify responses by H. akashiwo to commonly encountered nitrogen sources. Gene expression. The expression of NR, GS, and GOGAT was normalized to the transcript abundance of GAP (Figs. 2 and S1). NR expression in the NO-spiked samples was significantly higher than the Control for each experiment at 15 min ( Fig. 2A). Mean NR expression was highest in Experiment 1, and lowest in Experiment 3. At 60 min, the relative expression of NR remained significantly higher than Controls for Experiments 2 and 3 ( Fig. 2B), but there were no significant differences between Control and NO treatment for Experiment 1 at 60 min. The large standard deviation between biological replicates at this time point was likely due to the highly dynamic gene expression activity across biological replicates in Experiment 1. A comparison between time points indicates that the expression of NR decreased over time for Experiment 1 and increased over time for Experiments 2 and 3.
GS expression in the NO-spiked samples was not significantly different than the Controls for any experiment at 15 min, but was significantly lower than the Controls in Experiments 2 and 3 at 60 min (Fig. 2C, D). Similar to NR expression, GS expression was the highest in Experiment 1, and lowest in Experiment 3 at 15 min, while at 60 min, the mean GS expression was highest in Experiment 2 and lowest in Experiment 3. A comparison between time points indicates that the expression of GS relative to the Control decreased over time for all three experiments.
GOGAT expression in the NO-spiked samples at each time point was significantly higher than the Control in Experiments 2 and 3, but not significantly different from the Control in Experiment 1 (Fig. 2E, F). GOGAT expression in NO treatments at 15 min was similar across all three experiments. At 60 min, the mean expression of GOGAT was highest in Experiment 3 and lowest in Experiment 1. A comparison between time points indicates that the expression of GOGAT relative to Controls increased over time for all three Experiments (Fig. 2E, F).
Expression of NR, GS and GOGAT in nitrate-and ammonium-spiked treatments are shown in Supplementary  Fig. S1 (available online). The amount of added nitrate and ammonium (10% of ambient) differed by treatment and by experiment, so that direct comparisons should be viewed with caution. Instead, the nitrate and ammonium treatments provided a qualitative assessment of H. akashiwo's response to more commonly encountered nitrogen sources, and to provide context for its response to NO addition. NR and GS expression patterns and magnitude of expression relative to Controls when spiked with nitrate at both 15 and 60 min were similar to those of NO treatments. As expected, addition of ammonium inhibited NR expression relative to Controls at 15 min for all three experiments, while GS expression patterns when spiked with ammonium were similar to those of NO treatments at both time points. GOGAT expression at 15 min also followed the pattern of response observed for NO treatments. At 60 min, however, GOGAT expression for nitrate-spiked samples was lowest in Experiment 3, and lowest in Experiment 1 when spiked with ammonium.
Nitrate reductase enzyme assay. NR activity, measured at 2 h after spiking with NO, was up to 12 times higher than Controls, with no significant differences between NR activity between experiments (Fig. 3). NR activity was significantly higher than Controls in Experiments 1 and 3, but no significant differences were detected between Control and NO treatment in Experiment 2.
As with the gene expression analysis, direct comparisons between treatments or between experiments were not possible due to the differences in nitrogen added (10% ambient) for ammonium and nitrate. However, NR activity for nitrate-and ammonium-spiked cultures followed a pattern similar to NO treatments with highest activity in Experiment 1 and lowest activity in Experiment 2 (see Supplementary Fig. S2  www.nature.com/scientificreports/ Nitrogen assimilation ( 15 N). 15 N uptake and assimilation was analyzed at 4 (T4) and 24 (T24) hours after spiking with 15 N-labeled N. At T4, the 15 N uptake rate (Fig. 4A) of the NO-spiked samples was significantly higher in Experiments 2 and 3 compared to Experiment 1 (p value < 0.05). Likewise, the 15 N uptake per biomass ( Fig. 4C) for NO-spiked samples was significantly higher in Experiments 2 and 3 compared to Experiment 1 at T4 (p value < 0.05). The uptake rate of 15 N at T24 was significantly different between all three experiments (Fig. 4B). The highest 15 N uptake rate in NO-spiked samples occurred in Experiment 3 and the lowest was in Experiment 1. 15 N-labelled NO uptake per biomass was highest in Experiment 3 and lowest in Experiment 1 at T24 (Fig. 4D). 15 N uptake per biomass for NO-spiked samples was not significantly different between Experiments 2 and 3 (p value < 0.05).

Discussion
Stewart and Coyne 9 described a unique enzyme, NR2-2/2HbN (NR2) in Heterosigma akashiwo, that includes a HbN domain, potentially coupling nitric oxide dioxygenase (NOD) with nitrate reductase (NR) activity to assimilate NO. The ability to use NO as a nitrogen source has also been demonstrated for this species 10,11 , which may provide H. akashiwo with an advantage over other algae in regions where NO is available. This study investigated the effects of competing nitrogen types (nitrate, ammonium or an equal mix of nitrate and ammonium) on the ability of H. akashiwo to use NO. Transcript abundance for key genes involved in nitrate reduction (NR) and ammonium assimilation into amino acids (GS and GOGAT ), as well as NR activity and 15 NO uptake rate and assimilation into biomass were measured. Overall this research demonstrated that NO stimulated transcription of NR and GOGAT , as well as NR activity, resulting in incorporation of 15 NO into biomass by this alga. Coyne 28 showed that NR is constitutively expressed in H. akashiwo and upregulated by addition of nitrate. NR transcripts were detected under nitrogen starvation as well as in the presence of ammonium. Results presented Nitrogen-spiked controls received 15 N-labelled nitrate or ammonium at 10% of ambient concentrations (see Table 1), and the NO treatment was spiked with 100 µM 15 N-labelled S-nitroso-N-acetylpenicillamine (SNAP) to generate 15 (Fig. 2). Mechanisms involved in nitrogen uptake differ depending on nitrogen type: Nitrate and ammonium are moved into cells through inducible transporters 29 , while NO rapidly diffuses into cells 30 . Indeed, the highest expression of NR was measured at 15 min in cultures acclimated to growth on nitrate ( Fig. 2A). Even when acclimated to ammonium as a sole source of nitrogen (Experiment 3), however, NR transcript abundance was significantly higher than Controls within 15 min after spiking with NO. Prior research showed that exposure to NO has a variable effect on NR transcription in plants and other algal species, with reports of increased transcript levels 31 , inhibition 26 or no changes in transcript levels 32 . The mechanism for upregulation of NR expression by NO in H. akashiwo is not clear, but one possibility is that nitrate generated by NOD activity of constitutively expressed NR2 may have been sufficient to induce upregulation of NR. Comparison of NO spiked subcultures over time also supports the hypothesis that NR expression in response to NO is governed by regulatory mechanisms similar to cultures exposed to nitrate (see Supplementary Fig. S1 online). Interestingly, the magnitude of difference in the relative expression of NR in NO spiked subcultures decreased over time in cultures grown on nitrate alone (Experiment 1), indicating potential feedback inhibition from downstream products 33,34 . Glutamine, for example, has been shown to inhibit transcription of NR in Nicotiana plumbaginifolia 35 , while glutamine-derived ammonium downregulated NR in Dunaliella viridis 36 .
The increase in expression of NR after 60 min in NO-spiked subcultures acclimated to growth with ammonium (Experiment 3) or a mix of ammonium and nitrate (Experiment 2) may be due to alleviation of ammonium repression on transcriptional activity. In previous research, Coyne 28 showed that NR transcripts increased after nitrate was added to H. akashiwo cultures that were grown with ammonium, suggesting that nitrate: ammonium concentrations play a role in regulating expression 28 . Likewise, nitrate produced from NO through NOD activity  www.nature.com/scientificreports/ of NR2 may have increased intracellular nitrate: ammonium over time to overcome ammonium inhibition on NR transcription. Unlike NR, transcription of the cytosolic form of GS examined here did not immediately respond after exposure of the culture to nitric oxide. The activity of cytosolic GS coordinates nitrogen and carbon use efficiency and plays a major role in the assimilation of inorganic nitrogen as well as nitrogen recycling within the cell 37 . In diatoms, transcripts of cytosolic GS increased with the onset of darkness 38 , suggesting that it may also participate in dark assimilation of nitrogen. Here, expression of cytosolic GS in H. akashiwo at 15 min after spiking with NO was not significantly different from Controls, regardless of ambient nitrate or ammonium concentrations (Fig. 2C). The subsequent decrease in GS transcript abundance for NO-spiked cultures at 60 min compared to Controls for all three experiments suggests feedback inhibition from products of N assimilation (Fig. 2D). While little is known about the regulation of GS transcription in eukaryotic algal species, results of this study are consistent with research on plants and bacteria, indicating that transcription responds to cellular nitrogen status and is downregulated in response to feedback inhibition by accumulation of organic nitrogen products within the cell.
Results of this study showed that GOGAT expression was upregulated by NO addition in cultures acclimated to growth on ammonium (Experiment 3) or ammonium and nitrate (Experiment 2; Fig. 2E, F). It has been shown that expression of GOGAT is responsive to ammonium concentrations 39,40 , so that the increase in GOGAT expression over time in the present study was likely due to accumulation of downstream products of nitrate reduction, including ammonium and glutamine 39 . However, there is little supporting evidence of GOGAT regulation in microalgae and additional studies are required to fully understand mechanisms involved in regulation of both GS and GOGAT expression in these species.
Gene expression provides insight on the transcriptional response to nitrogen input but assimilation of nitrogen is also controlled post-transcriptionally, through mechanisms regulating the activity of the enzymes. The effect of NO on the activities of enzymes in nitrogen assimilation pathways has been studied in only a few model species. In Chlamydomonas reinhardtii, NR activity as well as the uptake of ammonium and nitrate were inhibited by NO 41 . Inhibition of NR in C. reinhardtii was mediated by truncated hemoglobins (THBs) with homology with the HbN domain of H. akashiwo NR2 24 . Although NR activity in H. akashiwo was previously found to be inhibited by ammonium 42 , the observed increase in NR activity with addition of NO in the current study was not significantly affected by the presence of ammonium (Fig. 3). It should be noted that NOD activity of NR2 was not measured here, and NR activity was used as a proxy for dioxygenase activity of NR2, converting NO to nitrate. Overall, results of the NR assay indicate that H. akashiwo responds to NO as it would to nitrate 28 , suggesting that increasing NO led to an increase in nitrate: ammonium concentrations to overcome repression of NR activity by ammonium.
While not measured here, less is known about the effects of nitrogen type on GS and GOGAT enzyme activities of algal species. Regulation of GS has been detailed primarily in plants 37 and bacteria, including cyanobacteria 43 , where a broad array of N-containing biomolecules indicative of cellular N status participate in cumulative feedback inhibition of GS activity. The study of dinoflagellate Akashiwo sanguinea showed that GS activity was highest when grown in ammonium, but was still active in the presence of nitrate, and steadily increased until high glutamate concentrations were reached 44 . A study of phytoplankton in the Taiwan strait showed a positive relationship between GS activity and ammonium but noted that this relationship was not maintained in unialgal culture studies of Emiliania huxleyi or Dunaliella primolecta 45 . Information on the induction and repression of GOGAT activity in eukaryotic algae is also limited. As with NR, GOGAT activity is likely to be modulated by the presence of substrates (glutamine and 2-oxoglutarate) and products. The present study indicated a trend toward increasing GOGAT expression with time after spiking with NO (Fig. 2E, F), suggesting a similar increase in substrate and GOGAT activity. An increase in GOGAT activity with increased transcription is also supported by the incorporation of 15 NO into cellular nitrogen as discussed below (Fig. 4).
The increased incorporation of 15 NO into biomass by cultures acclimated to growth on ammonium (Experiments 2 and 3; Fig. 4) is consistent with results reported previously for this strain of Heterosigma, showing a preference for nitrate over ammonium for this species 6 . In addition, the lower NO uptake rate and assimilation in the presence of nitrate (Experiment 1) compared to cultures with ammonium present may also be due to competition at the active site on NR. Nitrate binds to the molybdenum-molybdopterin cofactor, Mo-MPT, on NR 46 . Unlabeled nitrate present in the growth medium for Experiment 1 (see Fig. 1) and 15 N-labelled nitrate produced by the conversion of 15 NO to nitrate may compete for the active site of NR, resulting in the reduced uptake rate and lower amount of NO assimilation in the presence of nitrate compared to Experiments 2 and 3, where unlabeled nitrate was lower or absent altogether.
Diffusion may play a role in reduced uptake rate of the NO at 24 h compared to 4 h (Fig. 4). Since NO rapidly diffuses into the cell 29,30 , the abundance of NO within the cell available for assimilation would be greatest immediately after spiking cultures. As 15 NO was depleted over time, the ambient, unlabeled nitrogen in the form of either nitrate or ammonium would continue to be transported into the cell and assimilated into biomass, effectively diluting the signal from 15 NO.

Conclusions
Results of this investigation do not support the hypothesis that uptake and assimilation of NO by H. akashiwo is inhibited by ammonium. Indeed, NO upregulated NR expression and resulted in an increase in NR activity regardless of the presence of other inorganic nitrogen sources, with an increase in 15 NO uptake and assimilation in the presence of ammonium. Common regulatory mechanisms, such as accumulation of substrate and negative feedback inhibition by downstream products of nitrogen assimilation, may have affected the transcriptional and other regulatory responses to NO in a manner similar to that expected from nitrate. www.nature.com/scientificreports/ A better understanding of factors that stimulate the formation of algal blooms will improve management of coastal ecosystems and inform the development of HAB prevention and mitigation strategies. The capacity to use NO as an alternate nitrogen source may provide H. akashiwo with an advantage over other species in areas with high concentrations of NO, such as coastal environments, or when other nitrogen sources are scarce. This study suggests that NO availability should be considered a factor in promoting toxic blooms of H. akashiwo.

Methods
H. akashiwo was acclimated to growth on nitrate, ammonium, or a 50/50 mix of nitrate and ammonium in continuous cultures. Subcultures of each biological replicate were then spiked with NO and evaluated for gene expression, nitrate reductase enzyme activity and the uptake and assimilation of NO into biomass.
Chemicals. 15  Experimental design. Three experiments were conducted with different nitrate to ammonium ratios, while keeping total nitrogen concentrations constant (Fig. 1). Experiment 1 cultures were grown in modified ESAW with 100 µM nitrate. Experiment 2 cultures were grown in modified ESAW with 50 µM nitrate and 50 µM ammonium. Experiment 3 cultures were grown in modified ESAW without nitrate and with 100 µM ammonium. Cultures were dosed with Kanamycin (50 µg/mL) to prevent uptake and incorporation of 15 N-labelled substrate by bacteria 50 . Subcultures were created at 4 h after the start of the light cycle resulting in 4 replicates of 4 treatments for a total of 16 subcultures.
To verify responses to more commonly encountered nitrogen sources, the treatment subcultures were spiked with 15 N-labeled nitrogen equal to 10% of ambient nitrogen source. A fourth subculture was treated with 15 N-labeled SNAP at a concentration equal to total nitrogen concentrations of the continuous cultures (Table 1)  Primers for the cytosolic form of glutamine synthetase (GS) and glutamate synthase (GOGAT ) were designed using the "Primer-BLAST" function in Basic Local Alignment Search Tool (BLAST) 51 from National Center for Biotechnology Information (NCBI). Primers were designed from sequences in the 20130911 combined assembly for H. akashiwo CCMP2393 transcriptome, downloaded from the Moore Foundation Marine Microbial Eukaryote Transcriptome Sequencing Program 52 . PCR was conducted on cDNA with each primer pair and analyzed for the correct size fragment using gel electrophoresis. Primers were then optimized by qPCR and dissociation curves were evaluated to confirm specificity of primers.
Transcript abundance was measured by qPCR for GAP, NR, GS and GOGAT using an ABI 7500 Real-Time PCR System (Thermo Fisher Scientific). Diluted cDNA was used as template in triplicate 10 µL reactions. Each reaction consisted of 1 µL of template cDNA, 5 µL of SYBR Select Master Mix (Thermo Fisher Scientific), 2 µL sterile water, 1 µL each of forward and reverse primers ( Table 2). qPCR cycling parameters were as follows: 50 °C for 2 min and 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s, 60 °C for 30 s and 72 °C for 1 min. A dissociation stage was added to assess the specificity of amplification. Average transcript abundance was evaluated by linear regression analysis of triplicate reactions. Transcript abundance for NR, GS and GOGAT was then normalized to GAP 9,28 .
Nitrate reductase activity. Samples were collected from subcultures by centrifugation at two hours after spiking. The supernatant was discarded and the cell pellet was immediately frozen in liquid nitrogen. Samples were stored at − 80 °C until analysis. The frozen cell pellets were resuspended in 200 mM KPi extraction buffer (27.2 g L −1 KH 2 PO 4 , 10 g L −1 KOH, adjusted to pH 7.9) on ice. The resuspended pellet was homogenized by sonication, and centrifuged to clarify at 4 °C. The supernatants were immediately assayed as follows: triplicate 100 µL aliquots of extract were each mixed with 100 µL of 500 mM KPi assay buffer (pH 7.9) and 25 µL of 2 mM NADH (Sigma-Aldrich). KNO 3 (100 µM) was added to duplicate assays and sterile water was added to the third as a negative control reaction in place of KNO 3 . The reaction was incubated at room temperature for 15 min and 1 M zinc acetate was added to stop the reaction. The samples were centrifuged to pellet the zinc acetate and the supernatant was transferred to a new tube. The concentration of nitrite in the supernatant was measured colorimetrically by addition of 698 µM N-methylphenozonium methyl sulfate (Sigma-Aldrich). After incubation at room temperature for 20 min in the dark, 5 M HCl and 0.058 M sulfanilamide (Sigma-Aldrich) was added and the reaction was incubated at room temperature for another 5 min in the dark. 0.004 M N-(1-Naphthyl) ethylenediamine hydrochloride (Sigma-Aldrich) was then added and the reaction was incubated at room temperature for 10 min in the dark. Absorbance was measured at 543 nm using a Thermo Scientific Nanodrop 2000c (Thermo Fisher Scientific). NR activity was normalized to protein content of the cellular homogenate as determined using the Pierce BCA Protein Assay Kit (Pierce, Rockford, IL, US).

Nitrogen assimilation ( 15 N).
Samples were collected at 4 and 24 h after spiking for particulate 15 N analysis. Subsamples of each culture were vacuum filtered onto GF/F filters and dried at 60 °C for 24 h. Filters were folded into combusted tins and pelletized. Particulate 15 N in pelletized samples was measured by mass spectroscopy at Boston University (Experiment 1) or UC Davis (Experiments 2 and 3). Nitrogen uptake per biomass (V m , per hour) was calculated according to methods outlined in Dugdale and Wilkerson 53 and the uptake rate of nitrogen (Rho, µM/hr) was calculated according to Middelburg  Statistical analysis. Statistical analyses were performed in R using base functions and the "car" package.
Gene expression and NR assay data were first evaluated using the Q Test 55 and outliers (identified at 95% confi- www.nature.com/scientificreports/